Stabilized coenzyme solutions and their use for determining dehydrogenases or substrates thereof

ABSTRACT

Stabilized aqueous solution of a coenzyme for hydrogen-transferring enzymes characterized in that the solution contains NAD, NADP or a derivative thereof in an oxidized or reduced form and one or several organic compounds or salts thereof having a pKa value between 1.5 and 6.0 and/or a nitrogen compound of the general formula (I)  
                 
 
     in which the residues R 1 , R 2  and R 3  are the same or different and denote hydrogen, or a saturated or unsaturated alkyl or aryl group as well as the use of the solution to determine dehydrogenases in particular lactate dehydrogenase or substrates thereof.

[0001] The invention concerns stabilized aqueous solutions of a coenzymefor hydrogen-transferring enzymes and their use for determining acorresponding analyte (substrate) in a reduced form or for determiningthe enzyme activity of a corresponding dehydrogenase. The stabilizedsolution contains an organic compound or appropriate salts having a pKavalue between 1.5 and 6.0 and/or a hydroxylamine derivative.

[0002] The determination of enzyme activities (or substrateconcentrations), especially in blood serum or plasma, plays an importantrole in clinical chemical diagnostics. Test procedures are often usedfor this which are based on the reduction of nicotinamide adeninedinucleotide (“NAD”) or nicotinamide adenine dinucleotide phosphate(“NADP”) and photometric detection of the resulting change of theabsorption behaviour in the ultraviolet wavelength range (λ=334, 340 or365 nm). When suitable test conditions have been selected, this changeis linearly proportional to the enzyme activity (or substrateconcentration) to be determined.

[0003] Nowadays the methods described in Eur. J. Clin. Chem. Clin.Biochem. 31, 897 (1994) and Eur. J. Clin. Chem. Clin. Biochem. 32, 639(1994) are generally recommended for determining the enzyme activity offor example lactate dehydrogenase (LDH, E.C.1.1.1.27). The testprinciple involves the oxidation of lactate to pyruvate while a coenzymesuch as NAD or NADP is simultaneously reduced to NADH or NADPH. Such aconversion, in this case is for example catalysed by LDH, takes place inan alkaline medium (pH 9.4). As a result of this instability there is arelatively rapid increase in absorbance (the so-called reagent blank) inthe wavelength range for the measurement and hence the reagentcombination becomes unusable already after a short time (3 months) evenwhen stored in a refrigerator (2°to 8° C.). This is a particular problemfor the production of ready-to-use liquid reagents with a longshelf-life which are intended to enable the user to carry out analysesin the daily routine in a simple and reliable manner.

[0004] A method for stabilizing aqueous coenzymes using chelating agentsand azides is known from JP 84/82398. However, a disadvantage of thismethod is that it is necessary to add azide which is nowadays classifiedas cancerogenic and which, moreover, has an inhibitory effect on manyenzymes.

[0005] It is also known that coenzyme solutions can be stabilized byadding heavy metal salts, for example in the form of copper (II) ions,and thus prevent an increase of the reagent blank (DE 195 43 493 or EP 0804 610). However, degradation products may form during long storageperiods or at high storage temperatures (already above 10° C.) whichinhibit the dehydrogenase enzyme to be determined and thus result inmeasured values that are too low. A reagent that can be stabilized overa long period (3 months and more) of constant quality which thus, amongother things, does not require repeated calibrations is not available atpresent.

[0006] Hence the object of the present invention is to provide animproved stable liquid reagent containing a coenzyme forhydrogen-transferring enzymes which is suitable for determiningdehydrogenase activity or corresponding substrates.

[0007] The object is achieved by an aqueous solution which contains acoenzyme for hydrogen-transferring enzymes such as NAD, NADP or anappropriate derivative in an oxidized or reduced form (so-calledregenerating systems) and one or several organic compounds or saltsderived therefrom with a pKa value between 1.5 and 6.0 and/or a nitrogencompound of the general formula (I)

[0008] in which the residues R¹, R² and R³ are the same or different anddenote hydrogen, a saturated or unsaturated alkyl or aryl group.Suitable alkyl groups are in particular those which have one to tencarbon atoms. Furthermore the alkyl groups can be straight-chained orbranched. Suitable aryl groups according to the invention aresubstituted or unsubstituted phenyl groups which are optionally boundvia an alkenyl group which can have 1 to 8 carbon atoms. Nitrogencompounds of the said general formula (I) i.e. in particularhydroxylamine derivatives such as hydroxylamine, O- orN-alkylhydroxylamine having one to six carbon atoms orO-benzylhydroxylamine or salts derived therefrom such as sulfates,phosphates or ammonium salts have proven to be particularly suitableaccording to the invention. In addition suitable hydroxylaminederivatives are characterized by a complexing effect towards thedegradation products of the coenzyme.

[0009] In addition the stability of the solutions can be furtherimproved when the solution additionally contains a complexing agent i.e.a ligand which has two or more coordination positions. Bidentate ligandssuch as ethylene diamine and tetradentate or multidentate ligands suchas ethylenediamine-N,N,N,N-tetraacetic acid (EDTA) or appropriate saltsthereof, especially the disodium salt, crown ethers or cryptands haveproven to be advantageous. This corresponds to a concentration of thecomplexing agent of about 0.5 to 30 mM, preferably of 1.0 to 5.0 mm.

[0010] Organic compounds or salts derived therefrom that are addedaccording to the invention with a pKa value between ca. 1.5 and 6.0 areespecially organic acids which have a completing action and a bufferingaction in the pH range of 1.0 to 7.0 such as citric acid andwater-soluble salts derived therefrom.

[0011] The concentrations of the organic compounds, salts orhydroxylamine derivatives that are to be added according to theinvention can vary within wide limits i.e. between ca. 0.001 and 1.0 M.A concentration of ca. 5 to 200 mM has proven to be particularlysuitable for citric acid or citrate. In numerous cases ca. 50 mM citricacid or citrate already resulted in the desired effect. The preferredconcentration range for the hydroxylamine derivative according to theinvention that is to be added in addition to or in the absence of anorganic compound with a suitable pKa value is between about 2 and 300mM. The pH value of the stabilized aqueous solution can be between 1.0and 7.0, a pH value between ca. 2.0 and 4.0 or of ca. 3.0 having provento be particularly advantageous.

[0012] Moreover, it has proven to be particularly advantageous when thereagent containing NAD or NADP contains a hydroxylamine derivative andoptionally additionally a citrate salt and when boric acid or a boratesalt is additionally present in an optional further reagent that may benecessary to determine corresponding hydrogen-transferring analytes thatcontains in particular buffers necessary for the determination such asN-methylglucamine (MEG), substrates and optionally other auxiliarysubstances. The concentrations set forth above also apply to thisspecial embodiment. Furthermore, ca. 20 to 200 mM have proven to beparticularly advantageous for citrate or citric acid and ca. 10 to 150mM for the respective hydroxylamine derivative. A concentration range ofca. 50 to 200 mM has proven to be particularly suitable for the boricacid derivative which is preferably added to the substrate solution(so-called reagent 1) which does not contain NAD or NADP.

[0013] Substances which have a good buffer capacity between ca. pH 8.5and 10.0 such as the so-called Good buffers (tricine, bicine, TAPS,AMPSO, CHES, CAPSO, AMP, CAPS), carbonates of alkali metal ions, MEG,TRIS and phosphate buffer are basically suitable as buffers for thereagent containing substrate. Mixtures of the said buffer substanceshave also proven to be suitable for the solution according to theinvention. In addition it has proven to be advantageous when the bufferconcentration is between ca. 10 and 1000 mM, preferably between 200 and600 mM. Furthermore, the addition of boric acid or soluble salts andderivatives thereof to the alkaline buffer solution (reagent 1) whichprimarily determines the working pH value has proven to be advantageous.The concentration of suitable boric acid components is preferablybetween about ca. 50 and 200 mM, particularly preferably about 100 mM.Suitable coenzymes in the sense of the present invention are inparticular NAD and NADP, and also modified coenzymes such as thioNAD(P)or NHXDP (=nicotinamide hypoxanthine dinucleotide phosphate). Thecoenzymes can be present at a concentration of approximately 1.0 to 100mM in the reaction cuvette; a range of 5.0 to 15.0 mM is preferred.

[0014] The stabilized coenzyme solutions according to the invention arepreferably used in the form of aqueous solutions. Furthermore theready-to-use reagent is also stable over a wide time period as agranulate, powder mixture and as a lyophilisate. Thus no signs ofreagent decomposition whatsoever are found at temperatures of 2° to 8°C. within 15 months. Under stress i.e. at a temperature of ca. 35° C.for 2 weeks or treatment at ca. 42° C. for five days, it was shown thatthe solution containing one or several additives according to theinvention remained qualitatively unchanged i.e. stable.

[0015] A further subject matter of the invention is a method fordetermining a hydrogen-transferring analyte or a correspondingdehydrogenase in the presence of a hydrogen accepting coenzyme whereinthe coenzyme is present in a stabilized aqueous solution as describedabove.

[0016] The determination is carried out in particular in samples ofbiological origin such as whole blood, serum or plasma, or other humanor animal sources or in plant extracts. The sample can be prepared usingphysiological saline. In such a case a 0.9% NaCl solution isadvantageously used as a control value.

[0017] If it is intended to determine the enzyme activity of adehydrogenase such as a lactate dehydrogenase, a substrate solution e.g.a lactate solution in a substance (mixture) buffering at ca. pH 9.4 (37°C.) is used. In this case the substrate can be used in the usualconcentrations known to a person skilled in the art, preferably in arange of 40 to 80 mM.

[0018] In order to determine a hydrogen-transferring analyte such aslactate, the respective dehydrogenase, e.g. LDH, is added first in asubstance buffering between pH 8.5 and 10.0. Usually a dehydrogenasequantity of approximately 70 to 500 U/l, preferably of 110 to 220 U/l issufficient. The determination is usually carried out at ca. 37° C.

[0019] In addition to lactate which was described as an example, it isalso possible to similarly determine glutamate or ammonia, alcohol,glyceraldehyde-3-phosphate, glucose or other parameters that can beconverted by a suitable coenzyme-dependent dehydrogenase. This appliesin a corresponding manner to the determination of the enzyme activity ofsuch dehydrogenases.

[0020] A further subject matter of the invention is a so-called test kitfor carrying out the enzyme or analyte determination. The kit isessentially composed of two partial reagents. If it is used to determinethe activity of a dehydrogenase, the first reagent contains ahydrogen-transferring analyte (substrate) in a suitable system bufferingbetween pH 8.5 and 10.0. The second reagent has a coenzyme forhydrogen-transferring enzymes such as NAD or NADP and an organiccompound having a pKa value between 1.5 and 6.0 and/or a hydroxylaminederivative according to the invention. The second reagent canadditionally contain other auxiliary substances such as heavy metalsalts or a complexing agent. This applies correspondingly to thedetermination of an analyte or substrate such as lactate.

[0021] Abbreviations AMP = 2-amino-2-methyl-1-propanol AMPSO =3-[(1,1-dimethyl-2-hydroxyethyl)amino-2-hydroxy- propanesulfonic acidbicine = N,N-bis[2-hydroxyethyl]glycine CAPS =3-[cyclohexylamino]-1-propanesulfonic acid CAPSO =3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid CHES =2-[N-cyclohexylamino]ethanesulfonic acid MEG = N-methylglucamine TAPS =N-Tris[hydroxymethyl]methyl-3-aminopropane- sulfonic acid Tricine =N-Tris[hydroxymethyl]methylglycine TRIS =2-amino-2-(hydroxymethyl)-1,3-propanol

[0022] The invention is further elucidated by the following examples:

EXAMPLE 1

[0023] Reagent 1: 390 mmol/l N-methylglucamine pH 9.4 (37° C.); 60mmol/l lithium L-lactate.

[0024] Reagent 2: 60 mmol/l NAD(P) as a lyophilisate, powder mixture,granulate or aqueous solution.

[0025] Incubation temperature: 37±0.1° C.; measurement wavelength 340±2nm; path length 7 mm;

[0026] Preincubation: 5 minutes; lag phase: 2 minutes; measurement time:2 minutes.

[0027] Reagent 1=250 μl; reagent 2=50 μl; sample =7 μl NaCl solution(0.9% w/v).

[0028] The following determinations were carried out (IFCC: recognisedreference for the determination of LDH containing lactate, NAD/NADP andN-methylglucamine, pH 9.4; Eur. J. Clin. Chem. Biochem. vol. 32, p.639-655 (1994)), Table 1: TABLE 1 blank calibrator calibrator blankvalue (BV) signal signal value (BV) 5 days, -BV -BV unstressed 42° C.unstressed 5 days 42° C. reagent 1 reagent 2 [mA/min] [mA/min] [mA/min][mA/min] IFCC + IFCC 1.3 6.3 32.4 31.0 = 100 mmol/l 95.7% borate IFCCIFCC + 2.1 6.2 35.2 33.4 = 100 mmol/l 95.4% citrate pH 3.0 IFCC + IFCC +1.0 3.0 30.9 29.4 = 100 mmol/l 100 mmol/l 95.1% borate citrate pH 3.0IFCC IFCC + 0.9 3.2 34.4 33.8 = 50 mmol/l 98.3% hydroxylamine IFCC +IFCC + 0.7 1.8 31.1 30.5 = 100 mmol/l 50 mmol/l 98.0% boratehydroxylamine IFCC + IFCC + 0.7 1.2 29.8 30.3 = 100 mmol/l 50 mmol/l101.7% borate hydroxylamine + 100 mmol/l citrate pH 3.0 IFCC IFCC 1.511.5 35.7 33.7 = (prior 94.5% art)

[0029] Result: The inventive formulation containing appropriateadditives in the partial reagent 1 and/or partial reagent 2 shows aconsiderably improved blank value with an almost unchanged calibratorblank value compared to the IFCC reference method especially understress (5 days, 42° C.).

EXAMPLE 2

[0030] The initial solutions described in example 1 and thecorresponding procedures were used. Citrate and/or various hydroxylaminederivatives in different concentrations and combinations were added toreagent 2 (table 2). TABLE 2 blank calibrator calibrator blank value(BV) signal signal -BV value (BV) 5 days, -BV 5 days unstressed 42° C.unstressed 42° C. reagent 1 reagent 2 [mA/min[ [mA/min] [mA/min][mA/min] IFCC = IFCC = reference 0.1 10.7 35.7 32.9 = reference 92.1%IFCC IFCC + 20 mmol/l 0.8 2.5 31.6 31.2 = citrate + 50 mmol/l 98.7%hydroxylamine sulfate IFCC IFCC + 20 mmol/l 0.6 1.7 30.9 30.2 =citrate + 98.1% 50 mmol/l hydroxylamine phosphate IFCC IFCC + 20 mmol/l−0.9 −0.6 34.8 33.3 = citrate + 50 mmol/l 95.7% O-benzyl- hydroxylamineIFCC IFCC + 20 mmol/l −0.7 0.0 35.2 33.6 = citrate + 50 mmol/l 95.5%O-methyl- hydroxylamine IFCC IFCC + 20 mmol/l 4.9 11.1 34.6 34.6 =citrate + 50 mmol/l 100.0% N-methyl- hydroxylamine

[0031] Result: All compounds and salts added to the inventive reagent 2resulted in an improved recovery after stress (5 days, 42° C.) comparedto the IFCC reagent.

EXAMPLE 3

[0032] The recovery of the various isoenzymes was also demonstratedusing the formulation according to the invention. This must correspondto the recovery of the recognized IFCC recommendation (table 3).

[0033] The determinations were carried out using the IFCC reagentdescribed in example 1 (prior art) compared to a reagent according tothe invention. TABLE 3 activity activity activity activity activity iso-iso- iso- iso- iso- enzyme 1 enzyme 2 enzyme 3 enzyme 4 enzyme 5 reagent1 reagent 2 [U/l] [U/l] [U/l] [U/l] [U/l] IFCC IFCC 536 528 735 325 382IFCC + IFCC + 536 523 736 301 368 100 mmol/l 50 mmol/l boratehydroxylamine + 100 mmol/l citrate pH 3.0

[0034] Result: The recovery of the five LDH isoenzymes was demonstratedwith the reagent according to the invention. If the said modificationswere carried out on the formulation, it is possible to provide a liquidLDH reagent which remains stable during storage (>12 months) andtransport (even at temperatures >8° C.). The resulting advantages forthe user are obvious and are shown in the description of the invention.

1. Stabilized aqueous solution of a coenzyme for hydrogen-transferringenzymes characterized in that the solution contains NAD, NADP or aderivative thereof in an oxidized or reduced form and one or severalorganic compounds or salts thereof having a pKa value between 1.5 and6.0 and/or a nitrogen compound of the general formula (I)

in which the residues R¹, R² and R³ are the same or different and denotehydrogen, or a saturated or unsaturated alkyl or aryl group. 2.Stabilized solution as claimed in claim 1 , characterized in that itcontains an acid or a salt thereof with a buffering action in the pHrange of 1.0 to 7.0 as the organic compound.
 3. Stabilized solution asclaimed in claim 1 or 2 , characterized in that it contains citric acidor a citrate salt.
 4. Stabilized solution as claimed in claim 3 ,characterized in that it contains ca. 5 to 500 mM citric acid or acitrate salt.
 5. Stabilized solution as claimed in one of the claims 1to 4 , characterized in that the pH value of the solution is between 1.0and 7.0.
 6. Stabilized solution as claimed in one of the claims 1 to 5containing a hydroxyl, O- or N-alkyl-hydroxyl, O-benzylhydroxylamineand/or boric acid derivative.
 7. Stabilized solution as claimed in claim6 , characterized in that it contains a hydroxylamine derivative at aconcentration between 2 and 300 mM.
 8. Method for determining ahydrogen-transferring analyte or a corresponding dehydrogenase in thepresence of a hydrogen-accepting coenzyme characterized in that thecoenzyme is contained in a stabilized aqueous solution as claimed inclaims 1 to 7 .
 9. Method for the determination as claimed in claim 8 ,characterized in that the analyte lactate, glutamate, ammonia, alcohol,glyceraldehyde-3-phosphate or glucose is determined in the presence of alactate, glutamate, alcohol, glycerol-3-phosphate or glucosedehydrogenase.
 10. Method as claimed in one of the claims 8 or 9,wherein the pH value is in a range of 8.5 to 10.0 and the finalconcentration of citrate salt, boric acid and/or hydroxylaminederivative is in each case between 2 and 50 mM.
 11. Kit for determininga hydrogen-transferring analyte in a sample comprising the followingcomponents: a first reagent which contains a dehydrogenase in a suitablesystem buffering between pH 8.5 and 10.0 and a second reagent whichcontains a coenzyme for hydrogen-transferring enzymes and an organiccompound having a pKa value between 1.5 and 6.0 and/or a nitrogencompound of the general formula (I)

in which the residues R¹, R² and R³ are the same or different and denotehydrogen, or a saturated or unsaturated alkyl or aryl group.
 12. Kit fordetermining the enzyme activity of a dehydrogenase in a samplecomprising the following components: a first reagent which contains ahydrogen-transferring analyte in a suitable system buffering between pH8.5 and 10.0 and a second reagent which contains a coenzyme forhydrogen-transferring enzymes and an organic compound having a pKa valuebetween 2.0 and 4.0 and/or a hydroxylamine derivative of the generalformula (I) as claimed in claim 11 .
 13. Kit as claimed in claim 11 or12 , characterized in that the second reagent contains citric acid, acitrate salt, a boric acid and/or hydroxylamine derivative.
 14. Kit asclaimed in claim 11 or 12 , characterized in that the first reagentcontains a boric acid derivative and the second reagent contains citricacid, a citrate salt and/or a hydroxylamine derivative.
 15. Kit asclaimed in claim 11 to 14, characterized in that the second reagent hasa pH value between 1.0 and 7.0.
 16. Kit as claimed in one of the claims11 to 15 , characterized in that the second reagent has a pH value ofabout 3.0.
 17. Kit as claimed in one of the claims 11 to 16 ,characterized in that the first or second reagent contains approximately5 to 200 mM of a citrate salt, about 2 to 300 mM of a boric acidderivative and/or 2 to 300 mM of a hydroxylamine derivative.